Embryo modification and implantation

ABSTRACT

The present invention relates to constructs and methods used to enhance the attachment and implantation of an embryo. It is shown that modified glycolipids and glycolipid-attachment molecule constructs can be used to modify embryos, or localised to target tissues, to enhance interaction between the embryo and the target tissue, (typically the endometrium). The invention may advantageously be used to enhance implantation of embryos in the uterus, for example, in IVF treatments.

This invention relates to constructs and methods used to enhance theimplantation of an embryo into the uterus. In particular, the inventionrelates to modified embryos which have been modified by the insertioninto the cell membrane (or zona pellucida) of the embryo of constructswhich have a binding affinity for mucus or cell membrane surfaces, orenhance cellular interactions.

BACKGROUND

Each year 15% of couples seek medical advice because of difficultiesbecoming pregnant (WHO 1997). Sub-fertility is therefore currently oneof the most frequent health concerns facing the population aged 25-45years. For the past two decades, in vitro fertilisation (IVF) hasprovided an effective form of assistance for a large proportion of thesecouples. Indeed, IVF now accounts for 1.3% of all live births in Europe(Nygren et al. 2001) and 1.7% of all live births in Australasia (Hurstet al. 2001).

From the inception of routine IVF in 1978, pregnancy rates have risensteadily to levels considered normal for the fertile population(approximately 25% per attempt). The quest to break through thisphysiological barrier is driven by the significant financial andemotional cost for each IVF treatment for individuals.

Failure of embryos to implant into the lining of the uterus(endometrium) during an IVF treatment cycle is widely accepted by healthprofessionals as the most significant limiting factor to improvingsuccess rates. The scale of embryo wastage following transfer into IVFpatients is enormous, such that 80-85% of embryos fail to result in apregnancy (Blake et al 2002). Recent analysis of daily urine levels ofhuman chronic gonadotrophin (hCG) in women undergoing an IVF cycle,demonstrated that implantation was detected in as many as 60% of thecycles (Simon et a/1999). Of all embryos transferred in an IVF, 40% failto implant.

There are two broad reasons for failure of implantation followingreplacement of apparently viable embryos. The first involves intrinsicembryonic factors that reflect retarded development or deficiencies inthe health of the blastocyst itself and its ability to hatch (Gott et al1990, Plachot 1992, van Kooij et al 1996). The second relates toextrinsic factors that imply a lack of implantation receptivity in theendometrium (Edwards 1986, Yaron 1994). Moreover, successfulimplantation is dependent on the synchrony of embryonic development andendometrial maturation that is largely controlled by the ovarian hormonemilieu.

Recently it has become apparent that fertility drugs used for thesuper-ovulation of women undergoing IVF are predominantly responsiblefor the compromised implantation receptivity observed on both sides ofthe embryonic/maternal interface. Ertzeid and Storeng demonstrated thedetrimental effects of gonadotropins on implantation using a series ofcross-over embryo transfer experiments (Ertzeid et al. 2001). Embryosfrom super-ovulated and non-stimulated females were transferred toseparate uterine horns in the same super-ovulated or non-stimulatedpseudo-pregnant recipient mice. A significant decrease in implantationwas observed in the uterine horns receiving embryos from super-ovulateddonors and even more dramatically in both horns of super-ovulatedrecipients.

Highly elevated concentrations of estrogen result from ovarianstimulation in IVF. These are suspected to alter the cascade of hormonalevents and subsequent expression of cytokines that the oocytes, embryosand uterine endometrium would ordinarily be exposed to in anunstimulated menstrual cycle. Add to this the physiological challenge ofin vitro culture, largely devoid of growth factors, and it is notunexpected that IVF derived embryos might be compromised at the time ofimplantation.

Despite substantial advances in the recovery and maturation of multipleoccytes from unstimulated cycles, the practice of oocyte in vitromaturation (IVM) is as yet clinically unaccepted. With the prospect thatsuper-ovulation will remain the mainstay of IVF, other approaches toimproving implantation rates continue to be explored.

The development of physiological based culture media constituents hasgone some way to improving the development of embryos in culture for upto 6 days. This extended culture enables self-selection of the mostviable embryos for transfer, but as a consequence this approach has ahigh attrition rate of embryos. Co-culture of embryos on a mono- orbi-layer of support cells (e.g. endometrial cells) has also provided amethod for improving the development of embryos in culture presumablyvia the stimulus of growth factors. More directly the addition of avariety of growth factors to media has been explored and shown to be ofbenefit (Sjoblom et al. 2000).

Maintaining a receptive endometrium through administration of humanchorionic gonadotropin or progesterone has been practiced since theearly days of IVF. In fact only after additional progesterone supportwas given in the luteal phase of the cycle, did the world's first IVFpregnancies result. It has long been recognised that the elevatedestrogen profiles produced by the fertility drugs effectively advancethe endometrial tissue dating by approximately one day (Noyes et al.,1950; Pittaway et al. 1983; Garcia et al. 1984). Compound this with thefact that embryos are routinely transferred into the uterus at the 2-8cell stage (48-72 hrs prematurely to what occurs naturally) and it isclear that IVF results in an asynchronous environment for implantation.

Implantation of a hatched blastocyst is described as consisting of threephases:

-   -   a) apposition—where the embryo comes into initial physical        contact with the glycoconjugate coat of the endometrial        epithelium (called the glycocalyx).    -   b) adhesion—where the embryo undergoes cell to cell, and cell to        matrix binding with molecules derived from the apical cells on        the endometrium.    -   c) invasion—where the embryo penetrates through the epithelial        layer of the endometrium by intruding between cell junctions as        occurs in the human or by displacement of the cells found in        some animals (e.g. mice).

Super-ovulation has been postulated to alter electronegative propertiesof the glycocalyx and apical cell surface of the endometrium. In thisway; fertility drugs may reduce effective apposition and adhesion of atransferred embryo (Ronnberg et al. 1985).

At least two therapeutic approaches to improving implantation rates inIVF embryos have been practiced in humans. The first draws on theobservation that inclusion of the glycoaminoglycan, hyaluronan, in themedia containing embryos for transfer, results in a higher implantationrate than media devoid of this polysaccharide (Gardner et al. 1999). Theconcentration of hyaluronan increases in the uterus at the time ofimplantation in the mouse (Zorn et al. 1995) and is suggested tofacilitate implantation by a variety of means such as increasingcell-cell and cell-matrix adhesion and indirectly through promotion ofangiogenesis. Despite a lack of published trials in humans, hyaluronateis now present in a number of commercially available embryo transfermedia.

One therapy that has undergone clinical trials and is described in U.S.Pat. No. 6,196,965, is the use of a fibrin sealant. The firstexperiments with a fibrin sealant were carried out in 1981, and by 1988it had been proven safe to use in humans (Rodrigues et al. 1988).

U.S. Pat. No. 6,196,965 is based on the technique used in a randomisedclinical trial published in 1992 (Feichtinger et al. 1992). Embryos aretransferred in a catheter, sandwiched between small quantities ofthrombin/aprotinin and then fibrin. The results of the trialdemonstrated no significant difference in pregnancy rate between thecontrol and treatment group (546 patients), but a significant decreasein ectopic pregnancies in the fibrin sealant group.

The rationale and theoretical basis for the two therapeutic approachesdescribed above are different. Hyaluronate is added to transfer media inthe hope that it will induce a more physiologically receptiveenvironment for implantation. There is, however, an absence of directevidence at the molecular level proving this hypothesis. Fibrin sealanttherapy on the other hand, is used to encase the embryos in an adhesiveplug that will theoretically be glued onto the endometrium. Expulsion ofembryos from the uterine cavity by muscular contraction and avoidance ofectopic pregnancy was the predominant motivation for the fibrin sealantin the Feichtinger trial (Feichtinger et al. 1990), although otherinvestigators have hypothesised that fibrin would improve the adhesionphase of implanting embryos (Rodrigues et al. 1988).

In addition to the previously described therapeutic approaches, thespecification for international application no. PCT/US98/15124(published as WO 99/05255) describes the enhancement of implantation bycontacting the embryo with a lipid-modified adhesion molecule so as tomodify the development of the embryo. The technique of “proteinpainting” embryos with glycosylphosphatidylinositol (GPI) linked Qa-2proteins to increase the cell division rate is described.

Protein painting is a method for modifying the external antigens of cellmembranes without gene transfer. The method exploits the ability of GPIlinked proteins to spontaneously anchor to cell membrane via their lipidtails. The method described in the specification for internationalapplication no. PCT/US98/15124 (WO 99/05255) requires that a naturallyoccurring (or genetically altered) protein is inserted into an embryomembrane with an attached GPI lipid tail. Isolated GPI-anchored proteinsare stated as having an unusual capacity to reintegrate with acell-surface membrane. The molecules that can be used for modifying anembryo in this way are therefore confined to a rather limited group.

As described herein, the inventors have now found that embryos can bemodified with a range of selected synthesised molecules (modifiedglycolipids and glycolipid-attachment molecule constructs) and have theability to bind with mucus, and/or mucus components, and/or cellmembranes. The molecules are prepared exogenously by chemical orbiological processes.

Not only has the modification of embryos by the method of the inventionbeen successfully demonstrated in an in vitro culture system, butanimals have given birth to healthy offspring derived from modifiedembryos. Embryos prepared in accordance with the invention appear to bedevelopmentally indistinguishable from their unmodified counterparts.

It is an object of this invention to provide a modified embryo for theenhanced implantation of the embryo into the endometrium of an animal,or to at least provide the public with a useful choice.

STATEMENTS OF INVENTION

In a first aspect of the invention there is provided aglycolipid-inserted embryo for the preparation of an embryo modified forenhancing the implantation of the embryo into the endometrium of ananimal, where:

-   -   the glycolipid-inserted-embryo has an exogenously modified        glycolipid having lipid tails inserted into a cell membrane of        the embryo or into the zona pellucida of the embryo; and    -   the glycolipid has been modified to incorporate a binding part        wherein said binding part is adapted to enable binding to an        attachment molecule.

Preferably, the glycolipid has been modified to incorporate the bindingpart prior to the insertion of its lipid tails into the cell membranesof the embryo or into the zona pellucida of the embryo.

In a second aspect of the invention there is provided an embryo modifiedfor enhancing the implantation of the embryo into the endometrium of ananimal, where:

-   -   the embryo has an attachment molecule which is capable of        attaching to the endometrium; and    -   the attachment molecule is linked to the embryo by an        exogenously modified glycolipid having lipid tails inserted into        a cell membrane of the embryo or into the zona pellucida of the        embryo; and    -   the attachment molecule and the glycolipid have each been        modified to incorporate a binding part adapted to enable the        attachment molecule and the glycolipid to be bound together via        their respective binding parts either directly or through a        bridging molecule.

Preferably, the modification to the glycolipid is to the carbohydrateportion of the glycolipid.

Preferably the attachment molecule is a molecule known or adapted tointeract with the endometrium, mucus, mucin, or other endogenous orexogenously provided components of mucus. More preferably the attachmentmolecule is a known endometrial attachment molecule.

In one embodiment of the invention the binding interaction between theattachment molecule and the glycolipid are bound by way of non-covalentbinding interactions including ionic, van de Waals, water exclusion,electrostatic, hydrogen bonding and chelation binding or via covalentbonding.

In one embodiment of the invention the binding interaction between theattachment molecule and the glycolipid is avidin-biotin binding. In onepreferred embodiment the binding part of the glycolipid comprises biotinand the binding part of the attachment molecule comprises avidin. In analternative preferred embodiment the binding part of the glycolipidcomprises avidin and the binding part of the attachment moleculecomprises biotin.

In one embodiment of the invention the binding interaction between theattachment molecule and the glycolipid is through a bridging molecule.The bridging molecule may comprise avidin in the case of the bindingpart of both the attachment molecule and the glycolipid comprisingbiotin. Alternatively, in the case of the binding part of both theattachment molecule and the glycolipid comprising avidin, thebridging-molecule may comprise biotin.

In one embodiment of the invention the binding interaction between theattachment molecule and the glycolipid may be a chelation interaction.The binding parts of the attachment molecule and the glycolipid maytherefore be bridged by a chelated metal ion (e.g. Co²⁺, Ni²⁺ or Cu²⁺)and a poly-histidine recombinant protein. The chelator may be attachedcovalently or non-covalently (e.g. via biotin or avidin) to theglycolipid.

The glycolipid may be any glycolipid capable of inserting its lipidtails into the cell membranes of the embryo or into the zone pellucidaof the embryo such as phosphoglycerides or sphingolipids. The glycolipidmay be a natural molecule or a modified (e.g. biotinylated) glycolipid.Preferably the modified glycolipid is a biotinylated glycolipid eitherof the ganglioside class that contains sialic acid groups, or theneutral class that contains galactose.

The attachment molecule may be any molecule that has a binding affinityfor molecules on cell membranes (e.g. receptor sites and blood grouprelated antigens) including their mucus coat. Preferably the cellmembrane is endometrial. In particular, the attachment molecule ispreferably a protein, a peptide (such as poly L-lysine) a carbohydrate,an acyl group, a polymer, or an immunoglobulin such as immunoglobulin G(IgG) or a lectin. Alternatively, the attachment molecule may be asynthetic molecule (e.g. polyvinyl pyrrolidine, or an acyl group) whichreacts with molecules expressed on cell membranes or on the mucus layercovering the cell membrane. The attachment molecule can itself be aglycolipid or glycolipid conjugate.

In a third aspect of the invention there is provided a method ofpreparing the glycolipid-inserted-embryo of the first aspect of theinvention including the step:

-   -   contacting a glycolipid with an embryo, where the glycolipid has        been exogenously modified to incorporate a binding part, wherein        said binding part is adapted to enable binding to an attachment        molecule either directly or through a bridging molecule, so that        the lipid tails of the glycolipid insert into a cell membrane of        the embryo or into the zona pellucida of the embryo.

In a fourth aspect of the invention there is provided a method ofpreparing the modified embryo of the second aspect of the inventionincluding the steps;

-   -   contacting an attachment molecule with a glycolipid, where the        attachment molecule and the glycolipid have each been modified        to incorporate a binding part adapted to enable the attachment        molecule and the glycolipid to bind together via their        respective binding parts either directly or through a bridging        molecule to provide a glycolipid-attachment molecule construct;        and then    -   contacting the attachment molecule bound to the glycolipid        (glycolipid-attachment molecule construct) with an embryo so        that the lipid tails of the glycolipid insert into the cell        membranes of the embryo or into the zona pellucida of the        embryo:

Or including the steps:

-   -   contacting a glycolipid with an embryo, where the glycolipid has        been exogenously modified to incorporate a binding part adapted        to enable binding to an attachment molecule either directly or        through a bridging molecule, so that the lipid tails of the        glycolipid insert into a cell membrane of the embryo or into the        zona pellucida of the embryo; and then    -   contacting the glycolipid-inserted-embryo with an attachment        molecule, modified to incorporate a binding part wherein said        binding part is adapted to enable binding to the binding part of        the glycolipid either directly or through a bridging molecule.

Preferably the glycolipid, has been modified to incorporate a bindingpart comprising biotin and the attachment molecule has been modified toincorporate a binding part comprising avidin.

Alternatively, the glycolipid has been modified to incorporate a bindingpart comprising avidin and the attachment molecule has been modified toincorporate a binding part comprising biotin.

In the case of binding of the glycolipid to the attachment moleculethrough a bridging molecule, It is preferred that the bridging moleculecomprises avidin and that both the glycolipid and the attachmentmolecule have been modified to incorporate binding parts comprisingbiotin.

In a fifth aspect of the invention there is provided a method ofenhancing the implantation of an embryo into the endometrium of ananimal, preferably a human, or domesticated animal, comprising thesteps:

-   -   preparing a modified embryo according to the second aspect of        this invention, and    -   transferring the modified embryo to the uterus of the animal.

In one embodiment of the invention the modified embryo is prepared froma species, hybrid or variety of animal that is the same as the species,hybrid or variety of animal, to the uterus of which it is transferred.In an alternative embodiment, the species, hybrid or variety differ.

In a sixth aspect of the invention there is provided aglycolipid-attachment molecule construct when used for generating amodified embryo comprising a glycolipid modified to incorporate abinding part and an attachment molecule modified to incorporate abinding part wherein the respective binding parts are adapted to enablethe modified glycolipid and the modified attachment molecule to bindeach other either directly or indirectly through a bridging molecule.

In a seventh aspect of the invention there is provided a method ofenhancing the implantation of an embryo into the endometrium of ananimal including the steps of:

-   -   introducing a construct of the sixth aspect of the invention        into the uterus of the animal so that the construct becomes        localised to the endometrium; and then    -   transferring the embryo to the uterus of the animal.

In an eighth aspect the invention provides a kit for use in enhancingthe implantation of an embryo of an animal comprising one or morepreparations of a glycolipid-attachment molecule construct of the sixthaspect of the invention.

While the invention is broadly defined as above, those persons skilledin the art will appreciate that it is not limited thereto and that italso includes embodiments of which the following description providesexamples. In addition, the present invention will be better understoodfrom reference to the figures of the accompanying drawings.

FIGURES

FIG. 1—Schematic legend

FIG. 2—Schematic representation of natural glycolipid insertion. Anaturally occurring glycolipid (e.g. glycolipid A or Leb) is insertedinto a cell membrane (e.g. RBC, embryonic cell or endometrial cell).

FIG. 3—Schematic representation of biotinylated glycolipid insertion. Abiotinylated glycolipid (BioG) is inserted into a cell membrane (e.g.RBC, embryonic, endometrial cell).

FIG. 4 a—Schematic representation of terminal an attachment molecule(synthetic carbohydrate) and a glycolipid bound together. A biotinylatedglycolipid (e.g. BioG) is inserted into a cell membrane, conjugated toan avidin molecule and a biotinylated synthesised blood group A antigen(e.g. Atri-PAA) is attached.

FIG. 4 b—Schematic representation of an attachment molecule (IgG) and aglycolipid bound together. A biotinylated glycolipid (e.g. BioG) isinserted into a cell membrane, conjugated to an avidin molecule and abiotinylated IgG is attached.

FIG. 4 c—Schematic representation of an attachment molecule (lectin) anda glycolipid bound together. A biotinylated glycolipid (e.g. BioG) isinserted into a cell membrane, conjugated to an avidin molecule and abiotinylated lectin is attached.

FIG. 5—Schematic representation of the interaction between a modifiedembryo and a cell type. A BioG transformed cell of an embryo isconjugated to avidin and a biotinylated specific antibody (e.g.BiolgG^(A,B)). The resulting antibody transformed cell is then exposedto another cell type (RBC, embryo) expressing the corresponding antigen(e.g. blood group A or B) resulting in adhesion between the two celltypes.

FIG. 6—Schematic representation of the interaction between a modifiedembryo and a cell type. A BioG transformed cell of an embryo isconjugated to avidin and a biotinylated lectin (e.g. Bio-UE) isattached. The resulting lectin transformed cell is then exposed toanother cell type (e.g. endometrial) expressing the correspondingantigen resulting in adhesion between the two cell types.

FIG. 7—Schematic representation of BioG/Av/BiolgG insertion intoembryos. Adhesion is determined by reaction with antibody sensitisedcells (IgG bearing) via anti-human Ig.

FIG. 8 a—Schematic representation of one mechanism for demonstrating theadhesive protein model using BiolgG A or BioG/Av transformed mRBCs(murine red cells), and mouse embryos. Mouse embryos (right) will attachto the BiolgG^(A,B) transformed cells (eg RBCs) (left).

FIG. 8 b—Schematic representation of one mechanism for demonstrating theadhesive protein model using BiolgG^(A,B) or BioG/Av transformed mRBCs,and mouse embryos. Mouse embryos (right) that have been exposed toBiolgG^(A,B) (sensitised) will attach to the Bio/Av transformed cells(left).

FIG. 9 a—Schematic representation of BioUE adhesion model showing directinteraction between a BioUE transformed embryo (left) and H antigenbearing endometrial cells or red cells (right)

FIG. 9 b—Schematic representation of BioUE transformed embryos (left)reacting with group O secretor mucus as determined by inhibition ofreactivity of BioUE transformed embryos with group O red cells (humanred cells—hRBC).

DETAILED DESCRIPTION

The following description of this invention relates primarily to the useof biotin/avidin binding. It is important to note that othercombinations of attachment molecule and glycolipid modified toincorporate a binding part which allow high affinity conjugation (i.e.covalent or non-covalent bonding) between the attachment molecule andglycolipid are suitable.

Terms or expressions used to describe this invention are defined asfollows:

-   -   i. endometrium—The tissue lining the internal surface of the        uterus. It is this layer of epithelial cells and extracellular        matrix (i.e. mucus) that the implanting embryo comes into first        contact with. The epithelial and underlying stromal cell layer        cyclically thickens, secretes mucus and is shed from the body        under the hormonal influence of the menstrual cycle.    -    Attachment to the endometrium lining may be by interaction        between the attachment molecule and one or more components of        the endometrium, including membranes of the epithelial cells,        mucus, mucin components of the mucus, or an exogenously        introduced component of the mucus.    -   ii. zona pellucida—The glycoprotein coat that surrounds the        mammalian oocyte (egg) and embryo from the 1-cell to blastocyst        (6 day old) stage of development. Prior to embryo attachment and        implantation, the zona pellucida is shed from the embryo by a        number of mechanisms including proteolytic degradation. The zona        pellucida functions initially to prevent entry into the oocyte        by more than one sperm, then later to prevent premature adhesion        of the embryo before its arrival into the uterus.    -   iii. attachment molecule—Any carbohydrate or oligosaccharide,        glycolipid, glycoconjugate, protein or synthetic molecule that        can interact with one or more components of the targeted tissue        (e.g. endometrium) to localise the attachment molecule to the        tissue. Desirably the attachment molecule will interact with        endometrium and not the embryo.    -    The attachment molecule may be selected from natural or        synthetic carbohydrates or oligosaccharides, glycolipids,        glycoconjugates proteins, peptides, antibodies, lectins,        polymers such as polyvinyl pyrrolidine, and functionally        equivalent derivatives thereof.    -   iv. glycolipid—Any lipid-containing carbohydrate, including        phosphoglycerides (e.g. glycosylphosphatidylinositol) and        sphingolipids (e.g. glycosyl ceramides, cerebroside sulphate,        and gangliosides).    -   v. binding part—The portion of the attachment molecule or of the        glycolipid that interacts (or docks) with the attachment        molecule or glycolipid respectively, or with a bridging        molecule, to provide a non-covalent or covalent bond between the        binding part and the attachment molecule, glycolipid, or        bridging molecule, thereby providing a glycolipid-attachment        molecule construct.    -   vi. bridging molecule—a molecule that links the binding part of        the glycolipid with the binding part of the attachment molecule.        For example, avidin (interacting with biotin on either the        glycolipid or the attachment molecule), or a chelator        (interacting with a poly-histidine).    -   vii. biotin—Biotin is a water-soluble vitamin (H). It consists        of fused imidazolinone and thiophan rings with a pentanoate        side-chain attached to the latter. Biotin has an extremely high        affinity to bind the protein avidin via its imidazolidine ring.        The use of the term “biotin” in the description is intended to        be understood to include derivatives of biotin with functional        equivalence.    -   viii. avidin (Av)—Avidin derived from chicken egg white is a        glycoprotein with a molecular mass of 67 kDa. It contains four        identical sub-units, each bearing a biotin-binding site. The use        of the term “avidin” in the description is intended to be        understood to include derivatives of avidin with functional        equivalence.    -   ix. Chelation—Chelation is defined as the strong binding that        occurs between chelated metal ions and proteins. Certain        chemical groups called ligands, such as iminodiacetate and        nitrilotriacetate, form a stable metal coordination complex (or        metal chelate) with a divalent transition metal ion eg Ni²⁺,        Co²⁺ or Cu²⁺. Peptides containing poly-histidine residues        strongly bind to such a metal chelate by participation of        imidazole side-chains in chelation.    -   x. BioG (Biotinylated glycolipid)—Biotin coupled to a        glycolipid.    -   xi. BiolgG (Biotinylated Immunoglobulin G)—Biotin coupled to        immunoglobulin G. When the antibody has specificity this is        indicated as a superscript. For example BiolgG^(D),        BiolgG^(A,B), and BiolgG^(Lab) are biotinylated antibodies        directed against the D, ALe^(b), AB and Le^(b) antigens        respectively.    -   xii. Lectin—is a sugar-binding protein of non-immune origin that        agglutinates cells or reacts with glycoconjugates.

Glycolipids can insert into cell membranes without damaging cells. Theinvention provides for the insertion of synthetic molecules (includingexogenously prepared glycolipid-attachment molecule constructs) into theglycoprotein coat of early embryos (zona pellucida) and the lipidbi-layer membrane of embryo cells that are involved in embryoimplantation.

While this technology is applicable to embryo implantation in a widevariety of animals, it is most relevant to humans. However, thisinvention is not limited to human embryo modification and implantation.In particular inter species transfer, embryo modification andimplantation is contemplated.

One or several intercellular interactions can be targeted forimprovement using the technology of this invention. This may be a directadhesion mechanism, or other mitotic stimulus or cell recognitionevents. While the attachment molecule and glycolipid may be derived fromnatural or synthetic sources, the assembly of the attachment moleculeand the glycolipid is synthetic i.e. performed at least in partexogenously. The covalent or non-covalent, direct or indirect,attachment of the attachment molecule to the glycolipid may occur eitherbefore or after the insertion of the glycolipid into the cell membrane.

One combination that employs biotin/avidin binding is a biotinylatedglycolipid as the primary insertion molecule, an avidin bridgingmolecule, and a biotinylated attachment molecule (in this case anantibody or lectin or carbohydrate). The insertion process operates byexploiting the high binding affinity of avidin for biotinylatedmolecules, essentially forming a sandwich complex. Firstly, thebiotinylated glycolipid is inserted into the cell membrane to provide ananchor for the application of subsequent molecules. Secondly, theinserted cell membrane is treated with avidin that binds to thebiotinylated glycolipid. The final phase involves conjugation of theinserted molecules with the biotinylated endometrial adhesion molecule.To demonstrate this invention, the attachment molecules are theimmunoglobulin G antibody, a lectin (Ulex europeaus) and glycolipid.However, it must be emphasised that these molecules could be substitutedby any one of a variety of natural or synthetic molecules.

Immunoglobulin G and lectin were chosen for development of the inventionbecause of the ease in which molecular insertion and cell adhesionbetween two cell types can be confirmed using serological techniques.Preliminary development and proof of principle for each phase of theinvention was carried out using human RBCs. Essentially the red cellmembrane is a fluid membrane not too dissimilar to the embryo membrane,but much easier to obtain and handle. Thereafter, the insertiontechnique was tested on mouse embryos ranging from the 2-cell toblastocyst stage of development.

At each developmental phase, it was important to investigate thepotential risk of detrimental effects of the invention on embryonicdevelopment and maternal health. Initially, the morphologicaldevelopment of treated embryos was compared with control embryoscultured in vitro. The outcome of normal live births from transferredtreated embryos into recipient mice provides evidence of the safety ofthe invention. Finally, the ongoing reproductive performance of thetreated offspring proves that no lasting detrimental effects arepresent.

There are several steps in the practice and demonstration of the utilityof this invention;

-   -   Inserting natural glycolipids which may be potential        adhesion/communication molecules into embryo membranes (in        particular thorough carbohydrate-carbohydrate interactions, or        through carbohydrate-protein interactions);    -   Inserting modified (biotinylated) glycolipids in embryo        membranes as a mechanism to attach biotinylated molecules        through an avidin bridge;    -   Attaching IgG membrane/mucus adhesion molecules to embryo        membranes;    -   Attaching lectin membrane/mucus adhesion molecules to embryo        membranes;    -   Proving the embryo is unharmed by the processes above.

Adhesion of embryo's to cell membranes was proven initially byreactivity against red cells and secondarily against endometrial cells.For all intents and purposes red cells are equivalent to endometrialcells as they are of a similar size and are both fluid membranes. Insome instances red cells were considered as being “surrogate”endometrial cells. A serological technique known as rosetting (Indiveriet al 1979), was used to demonstrate the adhesive capacity of theembryo's which had been modified with adhesive proteins (eg antibodiesor lectins) with other cells. This was either done directly where theattached binding protein reacted with the corresponding antigen on thered/endometrial cells or through a bridge such as anti-IgG. In this wayit was possible to prove that not only had the adhesion molecule beensuccessfully inserted into the embryo, but that an artificial adhesionbetween two cell types had been created. For the purposes ofdemonstration the specificity of the antibodies selected were thosechosen to react with red cells, or for which glycolipid antigens existedwhich could be inserted into cells to make them express the desiredantigen. In the actual application of this technology red cell specificantibodies/lectins would be replaced with antibodies that detectantigens on the endometrial cells and/or mucins. The specificity of theantibodies or lectins which can be used is limited only by availability.

In order to insert molecules into cell membranes biotinylatedglycolipids (BioG; Example 1) and biotinylated antibodies (BiolgG;example 2) had to be prepared (when they could not be purchased). Theinsertion phenomenon using BioG and avidin concentrations were optimisedusing red cells (example 3).

Insertion Media

Stock glycolipids for insertion were prepared in a solvent free saline(see Example 4) to ensure protection from the reported detrimentaleffects of alcohols in sensitive embryonic cells (Lau et al. 1991). Thestock solution containing saline suspended (micelles) of glycolipids wasdiluted in various cell culture media or saline for insertionexperiments. The results in Example 5, are in agreement with otherinvestigators that the presence of serum, plasma or detergents isunnecessary for insertion to occur (Zhang et al. 1992). In contrast withprevious reports, the presence of albumin in the M2 media in Example 5,does not impede the insertion process. Therefore, the insertion solutionis effective in culture media with and without the presence of protein.Examples 6, 7 and 8 clearly demonstrate successful insertion ofglycolipids into endometrial cells and embryo's.

Inserting natural glycolipids which may be potentialadhesion/communication molecules into embryo membranes (in particularthrough carbohydrate-carbohydrate interactions, or throughcarbohydrate-protein interactions).

It is well established that cells can communicate through the lowavidity binding characteristics of carbohydrate-carbohydrateinteraction. These low affinity reactions are believed to be involved incellular communication and adhesion (Bovin, 1996; Hakomori 1996;Mikhalehik et al 2000; Wang et al 2001). Natural glycolipids can beadded to the surfaces of embryo and endometrial membranes, thusmodifying their carbohydrate expression (examples 6 and 7). Suchmodified cells may then potentially be available to react either withreactive carbohydrates expressed on the endometrial lining (membrane ormucus) or may react with carbohydrate reactive proteins expressed on theendometrial surfaces.

Inserting Modified (Biotinylated) Glycolipids in Embryo Membranes as aMechanism to Attach Biotinylated Molecules through an Avidin Bridge

Like the natural glycolipids (examples 6 and 7) biotinylated glycolipidsare able to be inserted into the embryo membranes including the zonapellucida (example 8).

The biotinylated ganglioside once inserted into the membrane is able tobe reacted with avidin, which can then pick up biotinylated molecules,thus modifying the surface of the embryo (examples 9-14).

Attaching IgG Membrane/Mucus Adhesion Molecules to Embryos Membranes

Several mechanisms were examined to show the attachment of IgG adhesionmolecules to cell membranes. These included direct attachment of anantibody which could react with the membrane of another cell for examplered cells (examples 9 and 10) and endometrial cells (example 12).Example 9 demonstrates the direct rosetting method, with an antibodyspecific to the red cell protein antigen D. Alternatively a multistageadhesion can be induced where some components are added to the embryoand others to the cell for adhesion. This can be seen in Example 10,where the carbohydrate specific IgG attachment molecule BiolgG^(A,B),was inserted into RBCs which adhered to embryos expressing the reactiveantigens. An alternative interaction was also demonstrated, whereembryos were coated with BiolgG^(A,B), and were shown to complex withRBCs inserted with BioG-avidin.

Additionally a bridging molecule such as anti-IgG could also be used tobridge IgG attached to both the membranes of the embryo and anothercell, in this case red cells (example 11) which it is desired the embryoattaches to. In this example, (example 11), human anti-D sensitised RBCswere used to demonstrate the adhesive properties of embryos insertedwith BiolgG, an immunoglobulin G with no specificity to any knownantigen. Addition of anti-IgG to the BiolgG embryos and anti-Dsensitised RBCs caused indirect resetting between the two cell types.Ideally an antigen, which is expressed on endometrial cells but isabsent on the embryo, would be an appropriate antibody target. In theabsence of easy availability of such a reagent and also to demonstrate afurther potential application, we inserted antigens into the endometrialcell membrane for which a a biotinylated antibody was available. Theseinserted antigens become integral parts of the cell membrane and as suchcan be considered part of the membrane (example 6). Blood group antigensLe^(b) and ALe^(b) were added to endometrial cells and the correspondingbiotinylated antibodies were attached to embryo's via BioG-avidin(example 12). The attachment of the endometrial cells to the embryo'sproves the mechanism of modified embryo induced adhesion. Additionallythis process opens up the opportunity to both insert molecules into theembryo and the recipient (e.g. lavage) to induce/enhance adhesionbetween the embryo and the recipient.

These various examples illustrate the use of IgG that can target eithercarbohydrate or protein antigens as attachment molecules for variouscell membrane attachment interactions.

Attaching Lectin Membrane/Mucus Adhesion Molecules to Embryos Membranes

Lectins are non-immunological carbohydrate binding proteins. In example13, the biotinylated lectin Ulex europaeus (BioUE) was inserted intoembryos to demonstrate a direct adhesive interaction with group C RBCsbearing the H type 2 carbohydrate antigen (specific antigen for UE).Additional the same phenomenon can be demonstrated with binding toendometrial cell culture (example 14)

Because the mucins will cover endometrial cells in utero the ability tomodify the embryo to react with antigens on mucus was also demonstrated.Lectins were used for this purpose but antibodies reactive with mucuswould be equally as applicable. In example 13 Ulex europaeus modifiedembryos were reacted with H type 2 containing mucus (obtained from humangroup O salivary secretions). In an inhibition assay the addition of Htype 2 bearing mucus inhibited red cell rosette formation, illustratingthat mucus had bound to the UE inserted embryos thus preventing thelectin reacting with red cells.

Proving the Embryo is Unharmed by the Processes Above.

An essential requirement of any implantation therapy is that it must notinduce any detrimental effects on the normal fetal growth of the treatedembryo, or the off spring, or the mother. Preliminary experiments withBioG inserted embryos showed no difference in morphology or zonahatching rate from control embryos during 5 days of in vitro culture(Example 15). Similarly, no difference from control embryos was noted(although not subjected to statistical analysis) between the pregnancy,live birth rate and normalcy of offspring in treated embryos (BioG,BioG/Av/BiolgG, ZI and ZF) when transferred into recipient mice (Example16 and Example 17). Ultimately, the ongoing fertility rate and secondgeneration pups of the offspring resulting from treated embryos wasapparently normal (Example 18).

EXAMPLES Example 1

Biotinylated gangliosides (BioG) were prepared using a modifiedprocedure described by Wilchek and Bayer (1987). The extraction andpurification of porcine gangliosides is carried out using establishedtechniques (Karlsson 1987, Ladisch et al. 1987, Ledeen et al. 1982).

-   -   1. Dried gangliosides purified from porcine brains, were        reconstituted in PBS with the aid of sonification.    -   2. The ganglioside sialic residues were oxidized by the addition        of sodium m-periodate.    -   3. The solution was subjected to 24 hr dialysis to remove the        resulting peroxide.    -   4. The oxidised ganglioside was incubated with biotin        amidocaproyl hydrazide (Sigma B-3770) for 1 hr.    -   5. The solution was subjected to further overnight dialysis in        water to remove excess biotin amidocaproyl hydrazide.    -   6. The resulting solution was dried via rota evaporation and        reconstituted in 50% methanol water. Further evaporation was        performed under nitrogen gas in a reduced pressure desiccator        overnight.

Example 2

Biotinylated immunoglobulin G was prepared using a method described byO'Shannessy 1990). Using similar procedures to those outlined in Example1, the IgG was oxidised with a periodate solution and incubated withbiotin amidocaproyl hydrazide.

Example 3

Optimum BioG insertion concentrations and conditions were established bylabelling the inserted BioG RBCs with avidin-FITC. The results areoutlined in Table 1. TABLE 1 Fluorescent signal of human RBCs insertedwith a range of BioG and labelled with avidin-FITC concentrations.Avidin-FITC BioG mg/ml mg/ml 10 5 2.5 0 0.90 ++++ ++++ +++ − 0.45 ++++++++ +++ − 0.33 ++++ ++++ +++ − 0.16 ++++ ++++ +++ − 0 − − − −

The optimum insertion concentration of BioG was 5 mg/ml. The minimumconcentration of avidin required for adequate detection of BioG at 5mg/ml concentration, was 0.16 mg/ml. The optimum minimum insertion timewas determined to be 1 hour as seen in Table 2. TABLE 2 Fluorescentsignal of human RBCs incubated with BioG for a variety of times, thenlabelled with avidin-FITC. Hours of incubation 0.25 0.5 0.75 1 1.5 2 4 626 +++ +++ +++ ++++ ++++ ++++ ++++ ++++ ++++

The amount of fluorescent signal score for the 2 hr incubation tubereduced from 4+ to 3+ after avidin labelling 5 days post insertion,suggesting minimal loss of inserted molecules over time.

Example 4

Stock glycolipids for insertion were prepared in solvent free saline toprotect sensitive cells such as embryos from solvent exposure duringinsertion treatment.

-   -   1. Purified dried glycolipids (e.g. Le^(b), A, or biotinylated        gangiloside) were dissolved in a glass tube with 50%        methanol/water to give a 10 mg/ml solution.    -   2. The solution was filtered with a 0.22 micron solvent        resistant filter into a sterile glass tube.    -   3. A 150 μl aliquot of the solution was marked on the side of        the glass tube to indicate the end point of evaporation. A        further 850 μl aliquot was placed in the glass tube to give a        total of 1 ml (containing 10 mg).    -   4. The tube was placed under a gentle stream of nitrogen gas in        a dry heat block at 50° C. until the meniscus was reduced to the        marked evaporation line.    -   5. The solution was made up to 200 μl with a balanced salt        solution of sterile PBS by adding 20 μl of 10× phosphate        buffered saline (PBS) and 30 μl of 18 mΩ water.    -   6. The final 50 mg/ml solution was aliquoted into sterile        microcentrifuge tubes and frozen at −70° C. or freeze dried (to        be later reconstituted with water).    -   7. Samples from the stock solution were then taken for dilution        in cell culture media for transformation experiments.

Example 5

The requirement for plasma or serum in the insertion media was shown notto be necessary. The ability for fluoresceinisothiocyanate-labelledavidin-(avidin-FITC Sigma A-2901) to bind to biotin formed the basis ofdetecting inserted BioG in RBCs when viewed under microscopefluorescence at 470 nm. In this study, a comparison in the degree offluorescent signal in avidin-FITC treated BioG human RBCs was carriedout for insertion solutions in a variety of tissue culture or serologymedia.

-   -   1. 5 μl of packed RBCs were mixed with 30 μl of 2 mg/ml BioG        (final conc. 12 μg/ml of packed RBCs) in one of the following        aqueous media for 2 hrs at 37° C. with frequent mixing. The        range of aqueous media included: Celpresol (CSL Biosciences,        Australia), M2 media mouse embryo handling media (Sigma M5910),        SQC mouse culture media (Vitrolife, Sweden), Medicult human        embryo culture media (Medicult Denmark) and PBS (made in-house).    -   2. The RBCs were washed 3× in saline by centrifugation and        incubated with 9.5 μl of avidin-FITC for 1 hr at 37° C.    -   3. The cells were washed 3× in saline and viewed under a        fluorescent microscope at 470 nm.

The concentrations and fluorescent microscopy results are outlined inTable 3. TABLE 3 Fluorescent signal of human RBCs inserted with BioG andlabelled with Avidin-FITC Celpresol PBS SQC Insertion albumin -albumin - albumin - M2 media free free free albumin 10% plasmaExperimental +++ ++ +++ ++++ ++ Negative − − − − − Controls

The presence of a clear fluorescent signal in both M2 and SQC cellculture media deemed them to appropriate for routine embryo insertionexperiments.

Example 6

The ability of natural glycolipids to insert into cell membranes wastested by inserting glycolipid A into endometrial cells. Insertion wasconfirmed by labelling with anti-A then by secondarily labelling withanti-mouse immunoglobulin conjugated to fluoresceinisothiocyanate(anti-mouse Ig-FITC) and detected by fluorescent microscopy.

A 5 million/ml heterogeneous solution of murine endometrial cells wasprepared by dissecting the uterine horn, scraping out the endometrialtissue, and incubating the tissue at 37° C. for 1.5 hrs in 500 μl of0.25% pronase and 1 ml of 0.5% collagenase. After incubation the cellswere washed and suspended in DMEM-F12 culture media

Glycolipid A was inserted and detected in endometrial cells using thefollowing method:

-   -   1. Freeze dried glycolipid A was resuspended in DMEM-F12 to give        a 10 mg/ml and a 1 mg/ml solution.    -   2. Three micro-centrifuge tubes were prepared each containing a        50 μl solution of 5 M/ml endometrial cells. The following        reagents were added to each micro-centrifuge tube a) 50 μl        glycolipid A (10 mg/ml), b) 50 μl glycolipid A (1 mg/ml) and c)        50 μl CMF(calcium magnesium free)-HBSS. The cells were incubated        overnight at room temperature.    -   3. After each treatment step the endometrial cells were washed 3        times by resuspending in M2 media and centrifuging at 2000 rpm        for 3 minutes. The washed cells were then resuspended in 50 μl        of M2 media.    -   4. Endometrial cells were subsequently reacted with anti-A by        adding 50 μl of anti-A murine monoclonal antibody to each        micro-centrifuge tube and incubating at room temperature for 30        minutes.    -   5. To test the presence by fluorescence 10 μl of mouse anti-Ig        FITC was added to each micro-centrifuge tube containing the        washed cells and incubated in dark conditions at room        temperature for 30 minutes.    -   6. Endometrial cells were mounted on glass slides and viewed        under a fluorescence microscope using a 470 nm filter and        photographed at 200-400× magnification.

7. The results of the experiment is outlined in Table 4 TABLE 4Fluorescent signal of murine endometrial cells inserted with blood groupA glycolipids. Insertion glycolipid Fluorescence Glycolipid A ++++ 10mg/ml Glycolipid A +++ 1 mg/ml Negative − Control

Example 7

The ability of natural glycolipids to insert into cell membranes wastested by inserting natural glycolipids A and Leb separately in murineembryos.

Glycolipid A and Le^(b) were inserted into the cell membranes of zonapellucida free (ZF) murine embryos from blastocyst to hatched blastocyststage. The insertion was confirmed by labelling with anti-A oranti-Le^(b) respectively, then by secondarily labelling with anti-mouseimmunoglobulin conjugated to fluoresceinisothiocyanate (anti-mouseIg-FITC) and detected under fluorescent microscopy.

Embryo insertion was performed in both M2 (Sigma M5910) and SQC(Vitrolife, Sweden) media using the following method:

-   -   1. Super-ovulated mouse embryos on day 3.5 post coitus were        obtained as described in Example 16.    -   2. Embryos from each mouse were stored in sterile        microcentrifuge tubes with M2 media.    -   3. Culture dishes were prepared with 3×50 μl micro-drops of        media overlaid with mineral oil.    -   4. Embryos with zonas intact (ZI) were placed in 0.25% pronase        (Sigma P8811) in CMF-HBSS media for 6 minutes at 37° C. until        the zona had disappeared. All embryos were zona free (ZF).    -   5. All embryos were washed 3 times in M2 media after each        treatment step by placing them into a fresh 100 μl drop of media        using a pulled glass capillary tube and syringe.    -   6. The following reagents were added to separate SQC        micro-drops: a) 50 μl Glycolipid A (10 mg/ml), b) 50 μl        Glycolipid Leb (5 mg/ml) and c) 50 μl M2 media. Equal numbers of        ZF embryos were placed in the micro-drops in a 5% CO₂, 37° C.        incubator for 120 minutes.    -   7. Embryos were subsequently cultured in a corresponding binding        antibody for each glycolipid. The following reagents were added        to separate micro-drops: a) 40 μl anti-A murine monoclonal, b)        40 μl anti-Leb murine monoclonal and c) 40 μl anti-A murine        monoclonal. The embryos from each group were placed in the SQC        micro-drops and returned to the 5% CO₂, 37° C. incubator for 30        minutes.    -   8. Embryos were transferred to a SQC micro-drop containing 20 μl        anti-mouse Ig-FITC and cultured in the drop for 1 hr in dark        culture conditions (in 5% CO₂, 37° C.).    -   9. Embryos were mounted on a glass microscope slide in a 2 μl        drop of media and overlaid with 2 μl of mineral oil.    -   10. The slides were viewed under a fluorescent microscope using        a 470 nm filter and photographed at 20-40× magnification.

The results of each experiments performed are outlined in Table 5. TABLE5 Fluorescent signal of murine embryo's inserted with glycolipids A andLe^(b) and labelled with anti-A or anti-Le^(b) respectively, thensecondarily labelled with anti-murine Ig FITC Insertion Le^(b)glycolipid A glycolipid glycolipid inserted inserted Experimental ++++++ Negative − − Controls

Example 8

Insertion of biotinylated gangliosides (BioG) into the cell membranes ofboth zona pellucida intact (ZI) and zona pellucida free (ZF) murineembryos from 2-cell stage through to hatched blastocyst stage wasconfirmed by a positive signal of avidin conjugated tofluoresceinisothiocyanate (avidin-FITC) detected under fluorescentmicroscopy. Some ZI embryos underwent zona removal post BioG insertionand pre avidin-FITC treatment to clearly visualise the degree of BioGinsertion in the cell membrane. Embryo insertion was performed in bothM2 (Sigma M5910) and SQC (Vitrolife, Sweden) media using the followingmethod:

-   -   1. Collection of super-ovulated mouse embryos on day 1.5 to day        3.5 post coitus was performed as described in Example 16.    -   2. Embryos from each mouse were split equally between control        and experimental groups where possible and transported from the        animal facility to laboratory in separate sterile        microcentrifuge tubes with M2 media.    -   3. A culture dish was prepared with 50 μl micro-drops of media        overlaid with mineral oil and the following reagents in separate        drops: a) 5 μl of BioG (50 mg/ml stock), and b) 5 μl of        avidin-FITC (1 mg/ml).    -   4. Embryos destined for ZF insertion treatment were placed in        0.5% pronase (Sigma P8811) in M2 media for 6 minutes at 37° C.        until the zona had disappeared.    -   5. All embryos were washed 3 times in M2 media after each        treatment step by placing them into a fresh 100 μl drop of media        using a pulled glass capillary tube and syringe.    -   6. ZF and ZI embryos were placed in the BioG micro-drop for 1-2        hours under appropriate culture conditions.    -   7. A group of ZI embryos were treated with 0.5% protease prior        to further treatment.    -   8. Embryos were subsequently cultured in the avidin-FITC drop        for 1 hr in dark culture conditions.    -   9. Embryos were mounted on a glass microscope slide in a 2 μl        drop of Citiflour (R1321, Agar Scientific, NZ) and overlaid with        2 μl of mineral oil, to replace the need for a cover-slip. A        felt tip marker was used to circle the location of the specimen.    -   10. The slides were viewed under a fluorescent microscope at        250-500× magnification using a 470 nm filter.

The results are outlined in Table 6. TABLE 6 Fluorescent signal emittedfrom embryos inserted with BioG and conjugated with avidin-FITC. Thedata represents the results of six experiments carried out on embryos atdifferent developmental stages from 2-cell to hatched blastocysts. Zonafree (ZF) embryos were treated with pronase, while hatched blastocystshad autonomously lost the zona. Some embryos were treated withavidin-FITC after further culture post BioG insertion. Unhatchedblastocysts and zona intact (ZI) embryos were BioG treated with the zonaretained Embryonic Expt stage Outline Result - fluorescent signal I2-Cell a) ZI M2 media controls a) nil embryos b) ZI M2 mediaexperimental b) cells +++ (zona ++++) freshly c) ZI SQC media controlsc) nil retrieved d) ZI SQC media experimental d) cells +++ (zona ++++)II 4-Cell cultured a) ZF controls a) nil from 2-Cell b) ZF experimentalb) +++ c) ZI controls c) nil d) ZI experimental d) +++ III late morulaa) ZF control embryos a) faint homogenous signal cultured from b) BioGthen pronase b) ++ to +++ 2-Cell c) pronase then BioG c) +++ to ++++Clear signal for polar body regardless of treatment IV unhatched and a)hatched controls a) nil (except in atretic cells) hatched b) hatchedexperimental b) ++ (stronger in atretic cells) blastocyst c) unhatchedcontrols c) nil except for atretic cells cultured from d) unhatchedexperimental d) cells +++ (zona ++++) 2-Cell e) arrested embryos e)cells +++ (zona nil) all treated with BioG then avidin- no difference inmorphology FITC 24 hrs later between control and experimental 24 hrspost BioG V blastocyst to a) ZI BioG and avidin-FITC a) + cells (zona ++to ++++) hatched day-6 blastocyst b) ZI BioG day-2 with further b) +cells (zona ++ to ++++) cultured from culture then avidin-FITC day-62-Cell VI unhatched BioG treatment 24 hours blastocysts previous asunhatched blastocysts then avidin-FITC treated as hatching blast.s a)pronase then avidin-FITC a) cells +++ to ++++ b) avidin-FITC thenpronase b) cells +++ to ++++ c) avidin-FITC no pronase c) cells + (zona+++ to ++++)

Example 9

Direct adhesion between an embryo and RBCs was demonstrated using thebiotinylated IgG specific for the protein antigen D (BiolgG^(D)). Inthis example, D+ve human RBCs were shown to positively rosette to mousezone free embryos inserted with BioG/Av/BiolgG^(D). No resettingoccurred on the surface of untreated mouse embryos nor those insertedwith BioG/Av only.

Mouse zone free embryos were inserted with BioG/Av and BiolgG^(D) usingthe following method:

-   -   1. Zona free day 3.5 mouse embryos were incubated at 37° C. for        1.5 hours in a 50 μl microdrop containing 5 μl of BioG (50        mg/ml), then washed 3× in M2 media.    -   2. The embryos underwent a 2^(nd) conjugation step where they        were exposed to 5 μl of avidin (1 mg/ml) in a 50 μl micro-drop        of media for 60 minutes at 37° C., and washed.    -   3. Finally, the embryos were incubated in a 50 μl microdrop        containing 25 μl of BiolgG^(D) (titre 1:1000) for 1 hour at 37°        C.    -   4. The embryos were washed 3× in M2 media and placed in a fresh        well of M2 media ready for rosetting with D+ve human RBCs.

The results are outlined in Table 7. TABLE 7 BioIgG^(D) transformedmouse embryo rosette experiment. D +ve human RECs adhere to mouseembryos that are transformed with BioIgG^(D) (Exp. group 3). Thisdemonstrates the ability for antibody transformed embryos to adhere to aprotein antigen on surrogate endometrial cells. No RBC adhesion wasobserved in either negative control groups. Experimental group 1 2 3Embryo configuration untreated BioG/Av BioG/Av/BioIgG^(D) RBC type D+veD+ve D+ve Rosetting nil nil ++

Example 10

Direct adhesion between an embryo and RBC was demonstrated using thebiotinylated IgG specific for the carbohydrate antigens A,B(BiolgG^(A,B)). In this example, two combinations of insertion weretested. In the first instance, BiolgG^(A,B) inserted mouse RBC's wereshown to rosette to mouse embryos that are known to express an antigenreactive with IgG^(A,B). Additionally, mouse RBC's inserted with BioGand avidin only, positively adhered to mouse embryos sensitised (coated)with BiolgG^(A,B).

Mouse RBCs were inserted with BioG/Av and BioG/Av/BiolgG^(A,B) using thefollowing method:

-   -   1. 5 μl of BioG (50 mg/ml) was added to 500 μl of 10% mouse RBCs        in PBS (final conc. 5 μg/μl of packed RBCs) and incubated on a        mixer at 37° C. for 1 hour. The cells were washed 3× in PBS and        resuspended to 500 μl.    -   2. 50 μl of 1 mg/ml avidin was added to the BioG inserted RBC        solution (final conc. avidin 0.1 mg/ml) and incubated on a mixer        at 37° C. for 1 hour. The cells were washed 3× in PBS and        resuspended to give a 10% solution of RBCs. These BioG/Av        inserted cells were used to react with embryo's sensitised with        BiolgG^(A,B)    -   3. A 100 μL aliquot of BioG/Av inserted RBCs was mixed with 50        μl of BiolgG^(A,B) (10 μg/10 μl of packed RBCs) and incubated on        a mixer at 37° C. for 1 hour. The cells were washed 3× with PBS        ready for the rosetting with the embryos. These        BioG/Av/BiolgG^(A,B) inserted cells were used to react directly        with embryos.

Mouse embryos were sensitised with IgG^(A,B) using the following method:

-   -   1. ZF and ZI day-3.5 embryos were incubated in a microdrop of 25        μl of M2 media and 25 μl of BiolgG^(A,B) (final conc. 0.1 mg/ml)        for 1 hour at room temperature.    -   2. These BiolgG^(A,B) sensitised embryos were washed 2× in M2        media and placed in a fresh well of M2 media ready for rosetting        by exposure to the BioG/Av inserted mouse RBCs.

The results are outlined in Table 8. TABLE 8 Embryo BioIgG^(A,B) rosetteexperiment. Group 1 ZF embryos rosette with BioG/Av/BioIgG^(A,B) mouseRBCs. Group 2 ZF and ZI embryos sensitised with BioIgG^(A,B) rosettewith BioG/Av transformed RBCs. Group 3 embryos represent a negativecontrol and fail to rosette with BioG/Av transformed RBCs. ExperimentalGroup 1 2 3 mRBCs BioG/Av/BioIgG^(A,B) BioG/Av BioG/Av configurationembryo untreated BioIgG^(A,B) treated untreated configuration ZF binding+++ +++ − ZI binding − ++ −A grade from nil to 4+ was allocated to each group of embryos:nil no binding+ <10 RBCs++ 10-20 RBCs++++ >50 RBCs

Example 11

The ability of modified embryos to adhere (through an immunologicalbridge) to antigens on other cell types was tested. In this example, theadhesion molecule was classified as biotinylated non-specific IgG andanti IgG [BiolgG+anti-IgG] which was conjugated in a third step to theinserted molecules BioG and avidin, on the cell membranes and zonapellucida of murine embryos. To confirm the complete insertion of thiscomplex, IgG sensitised RBCs were allowed to rosette. The IgG that isattached to the anti-D sensitised RBCs is used as an antigen for theadhesion molecule—thus the antibody coating on the cells essentiallyacts as a cell bound protein antigen. This model is considered anindirect demonstrative example of adhesion, because anti-IgG is requiredto complete the adhesive complex.

Insertion, conjugation and adhesion of IgG sensitised RBCs was carriedout as follows:

-   -   1. Anti-D sensitised RBCs were made by incubating 400 μl of        human serum containing human anti IgG with 200 μl of RhD+ve        human RBCs for 1 hr at 37° C. The RBCs were then washed in        Celpresol and made up to a 5% solution for the rosette        technique.    -   2. All embryos were retrieved from super-ovulated mice at the        2-cell stage and entered into the experiment either on the day        of retrieval or after 48 hours of cell culture in SQC media        (late morula to blastocysts stage).    -   3. BioG insertion was performed on both zona intact and zona        free embryos with either M2 or SQC used as the insertion media.    -   4. The embryos then underwent a 2^(nd) conjugation step where        they were exposed to, 5 μl of avidin (1 mg/ml) in a 50 μl of        micro-drop of media for 60 minutes at 37° C. in appropriate        culture conditions for each media type (i.e. CO₂ or        atmospheric).    -   5. The washed embryos underwent a 3rd conjugation with 5 μl of        BiolgG (1 mg/ml) in a micro-drop of media for 30 minutes at 37°        C., then washed.    -   6. The embryos were placed in a micro-drop consisting of 25 μl        of M2 media and 25 μl of monoclonal anti-IgG for 30 minutes at        22° C.    -   7. The treated and control embryos were washed and placed in        separate drops of M2 media. A stream of either 50% anti-D        sensitised RBCs or 50% untreated D+ve RBCs were gently blown        over the embryos using a pulled capillary pipette attached to a        syringe.

8. After 10 minutes at room temperature, the embryos were gentlytransferred to fresh media micro-drops using a wide bore capillarypipette (170 μm diameter) and assessed for RBC adherence under aninverted microscope at 250× magnification through the central plane offocus. A grade from nil to 4+ was allocated to each group of embryos:nil no binding + <10 RBCs ++ 10-20 RBCs ++++ >50 RBCs

The results are shown in Table 9. The adhesion of large quantities ofanti-D sensitised RBCs to embryos (2-cell to blastocysts) indicatespositive insertion of Bio/AV/BiolgG and demonstrates the ability oftransformed embryos to adhere. There was no difference in the adhesionscore between M2 and SQC insertion media. The adhesion score wasmoderately greater in the zona intact embryos than the zona freeembryos. TABLE 9 Adhesion scores of BioG/Av/BioIgG transformed embryoswhen exposed to either anti-D sensitised or untreated group D +ve RBCs.Experiment I compares the adhesion in zona intact 2-Cells when stepswere carried out in M2 or SQC media. Experiment II assesses adhesion inlate morula (LM) to blastocyst (blast) stage zona intact embryos.Experiment III compares adhesion in LM-blastocyst zona intact and zonafree. Zona Stage of free Embryo embryos Y/N treatment type RBCsAdherence Expt. I 2-Cell N BioG/Av/BioIgG D +ve − M2 media 2-Cell NBioG/Av/BioIgG anti-D +++ M2 media sensitised 2-Cell N BioG/Av/BioIgG D+ve − SQC media 2-Cell N BioG/Av/BioIgG anti-D +++ SQC media sensitisedExpt. II LM-Blast N BioG/Av/BioIgG anti-D ++++ sensitised LM-Blast NBioG/Av/BioIgG D +ve − LM-Blast N Control anti-D − untreated sensitisedLM-Blast N Control D +ve − untreated Expt. III LM-Blast N BioG/Av/BioIgGanti-D +++ sensitised LM-Blast N Control D +ve − untreated LM-Blast YBioG/Av/BioIgG anti-D ++ sensitised LM-Blast Y Control D +ve − untreated

Example 12

Carbohydrate antigens and anti-carbohydrate binding antibodies wereutilised to demonstrate adhesion between embryo and endometrial cells.In this example both embryo and endometrial cells were modified withcorresponding binding molecules (IgG antibodies directed againstcarbohydrate antigens and antibody reactive glycolipid antigens).

Two series of insertion were tested. In the first the biotinylatedantibody directed against the ALe^(b) antigen (BiolgG^(ALeb)) wasprepared (example 2) and inserted into embryo cell membranes via theBioG/avidin bridging mechanism while endometrial cell membranes weremodified with the corresponding glycolipid ALe^(b) antigen. In thesecond combination biotinylated antibody directed against the Le^(b)antigen (BiolgG^(Leb)) was inserted into the embryo cell membranes viathe BioG/avidin bridge while endometrial cell membranes were modifiedwith the Le^(b) glycolipid antigen.

Glycolipid modified endometrial cells were shown to adhere to theantibody modified embryos.

Murine endometrial cells were prepared as follows;

-   -   1. A 5-10 million/ml heterogeneous solution of murine        endometrial cells was prepared as described in example 6.    -   2. Three micro-centrifuge tubes each containing 50 μl of 5-10        million/ml endometrial cells were prepared. The following        reagents were added to separate tubes a) 50 μl ALe^(b)        glycolipid (5 mg/ml) b) 50 μl Le^(b) glycolipid (5 mg/ml) and c)        50 μl DMEM-F12. All cells were incubated overnight at room        temperature to allow the glycolipid molecules to insert.    -   3. The endometrial cells for the ALe^(b) experiment were treated        with a fluorescent stain by adding 10 μl of acridine        orange/ethidium bromide solution to a 50 μl of endometrial        cells. All cells were incubated in dark conditions at 37° C. for        30 minutes. This fluoro'chrome staining of the endometrial cells        prior to embryo contact assists in identifying endometrial cells        adhered to embryos by fluorescent microscopy.    -   4. The endometrial cells were washed 3 times by suspending in        CMF-HBSS and centrifuging at 2000 rpm for 3 minutes.

Mouse zona free embryos were inserted with BioG/Av and BiolgG^(ALeb) orBiolgG b using the following method:

-   -   1. Collection of super-ovulated mouse embryos on day 3.5 post        coitus was performed as described in Example 16.    -   2. Micro-drop culture dishes were prepared with 50 μl of M2        media overlaid with mineral oil.    -   3. Embryos from each mouse were placed in SQC media microdrops        and incubated in a 5% CO₂, 37° C. incubator overnight.    -   4. Embryos with zonas intact were placed in 0.25% pronase in        CMF-HBSS media for 6-8 minutes until the zona had disappeared.    -   5. All embryos were washed 3 times in M2 media after each        treatment step by placing into a fresh drop of M2 media using a        pulled glass capillary tube and syringe.    -   6. Zona free day 4.5 experimental mouse embryos were incubated        at 37° C. for 1.5 hours in a 50 μl SQC micro-drop containing 5        μl of BioG (50 mg/ml).    -   7. The experimental embryos underwent a second conjugation step        where they were exposed to 5 μl of avidin (1 mg/ml) in a 50 μl        micro-drop of media for 60 minutes at 37° C.    -   8. Finally, the experimental embryos were split into two groups.        Group 1 were placed in a 50 μl micro-drop containing 25 μl of        BiolgG^(ALeb) at 5 mg/ml. Group 2 in a 50 μl micro-drop        containing 25 μl of BiolgG^(Lab) at 5 mg/ml. Embryos were        incubated for 1 hour at 37° C.

BiolgG^(ALeb) and BiolgG^(Lab) transformed mouse embryos weresubsequently immersed in the corresponding modified endometrial cells totest for attachment in a two step process.

-   -   1. Micro-centrifuge tubes were prepared with the following: a)        acridine orange stained ALe^(b) glycolipid modified endometrial        cells into which was placed BioG/Av/BiolgG^(ALeb) inserted        embryos; b) unstained Le^(b) glycolipid modified endometrial        cells into which was placed BioG/Av/BiolgG^(Leb) inserted        embryos.    -   2. The tubes containing the endometrial cells and embryos were        gently mixed for 30 minutes at 37° C. Contents of each        micro-centrifuge tube were transferred to a 4 well culture        plate.    -   3. Embryos were carefully removed from the wells and mounted on        glass slides. Embryos were viewed under a fluorescence        microscope and photographed at 200-400× magnification.

The results are outlined in Table 10 TABLE 10 Degree of attachment ofALeb and Leb glycolipid inserted endometrial cells to murine embryo'sinserted with BioG/Av/BioIgGALeb and BioG/Av/BioIgGLeb respectively.Modifications to embryos and endometrial cells Embryo: BioG/Av/BioIgG^(Leb) Embryo: BioG/Av/BioIgG^(ALeb) Endometrial Endometrialcells: ALe^(b) + AcOr cells: Le^(b) Cell attachment ++ ++ observed inbright field Cell attachment +++ ND observed under fluorescenceAcOr = fluorochrome acridine orangeCell attachment scoring; + = 1-4 cells, ++ = 5-10 cells per embryo,+++ > 10 cells per embryoND = Not Done

Example 13

The adhesive properties of Ulex europaeus inserted mouse embryos, wasconfirmed by direct adhesion to group O human RBCs by resetting. Ulexeuropaeus is a lectin that binds specifically to the carbohydrateantigen H type 2 present on the surface of group O human RBCs and in themucus/saliva of group O individuals expressing the secretor phenotype.Adhesion of UE transformed embryos to secretor mucus was alsodemonstrated by the inhibition of rosetting with group O RBCs afterprior exposure to the mucus.

Insertion and conjugation of embryos with UE was conducted as follows:

-   -   1. The zona pellucidae were removed from embryos by incubating        in a 100 μl microdrop of 0.25% pronase in M2 media at 37° C. for        6 minutes, then washed 3× in M2 media.    -   2. Embryos were incubated in a 50 μl microdrop of SQC media        containing 5 μl of 50 mg/ml BioG (final conc. 0.2 mg/ml) for 1.5        hrs at 37° C.    -   3. The washed embryos were incubated in a 50 μl microdrop of SQC        media containing 5 μl of avidin 1 mg/ml (final 0.1 mg/ml) for 1        hr at 37° C., then washed 3× in M2 media.    -   4. Finally the embryos were incubated in a 50 μl microdrop of        SQC media containing 25 μl of BioUE 100 μg/ml (final conc. 50        μg/ml) for 40 minutes at 37° C. After washing 3× in M2 media,        the embryos were placed in a fresh drop of M2 media, in        preparation for RBC resetting.    -   5. Group 3 and group 4 BioUE inserted embryos were incubated for        30 minutes at RT in a 50 μl drop of a 1:10 dilution of secretor        and non-secretor mucus (respectively). The embryos were placed        in a fresh drop of M2 media without washing in preparation for        RBC resetting.    -   6. All untreated, treated and mucus pre-exposed groups of        embryos, had a stream of group O RBCs gently aspirated around        them. After 10 minutes incubation at RT, the embryos were        carefully transferred to a fresh drop of M2 and the degree of        RBC attachment was observed under an inverted microscope as        described in Example 11.

The results are outlined in Table 11. TABLE 11 Ulex europaeustransformed mouse embryo rosette and mucus inhibition experiment. GroupO (H type 2 bearing) RBCs adhere to mouse embryos that are transformedwith UE (Exp. group 2). This adhesion is inhibited by pre-exposure to Osecretor H type 2 bearing mucus (Exp. group 3 -) but not by Onon-secretor mucus (Exp. group 4) where the H type 2 antigen is absent.Experimental group 1 2 3 4 Embryo configuration untreated BioG/Av/BioUEBioG/Av/BioUE BioG/Av/BioUE Mucus incubation nil nil O secretor Onon-secretor RBC type O O O O Rosetting nil +++ nil +++Cell attachment scoring + = 1-4 cells, ++ = 5-10 cells per embryo, +++ >10 cell per embryo

Example 14

In this example both embryo and endometrial cells were modified withcorresponding binding molecules. The biotinylated lectin Ulex europaeus(BioUE) was inserted into embryo cell membranes via the BioG/avidinbridging mechanism (BioG/Av/BioUE). Endometrial cell membranes weremodified with glycolipid H type 2 and stained with pyronine Y.Fluorochrome staining of the endometrial cells prior to embryo adhesionassists in identification of bound endometrial cells when visualised byfluorescent microscopy.

Modified endometrial cells were shown to adhere to UE transformedembryos. In comparison, minimal attachment on the surface of untreatedmouse embryos was observed.

Endometrial cells were inserted with H type 2 glycolipid and stainedwith pyronine Y by the following method.

-   1. A 5-10 million/ml heterogeneous solution of murine endometrial    cells was prepared (as per example 6).-   2. Two microcentrifuge tubes were prepared each containing a 1 ml    solution of 5-10 M/ml endometrial cells. The cells were subsequently    centrifuged at 2000 rpm for 3 minutes before aliquoting the    supernatant to leave 5 μl of packed endometrial cells in each tube.    100 μl of H type 2 glycolipid extracted from human group O red cell    membranes (10 mg/ml) was added to the experimental group of    endometrial cells and 100 μl of DMEM-F12 media added to the control    group of endometrial cells. All cells were incubated overnight at    room temperature.-   3. After each treatment step the endometrial cells were washed 3    times by resuspending in CMF-HBSS media and centrifuging at 2000 rpm    for 3 minutes.-   4. Endometrial cells were treated with pyronine Y by adding 20 μl of    pyronine Y (15 μg/ml) to each microcentrifuge tube and incubating at    37° C. in dark conditions. The cells were thoroughly washed.

Mouse zona free embryos were inserted with BioG/Av and BioUE using thefollowing method:

-   -   1. Collection of super-ovulated mouse embryos on day 3.5 post        coitus was performed as described in example 16    -   2. Micro-drop culture dishes were prepared with 50 μl of SQC        media overlaid with mineral oil.    -   3. Embryos were placed in SQC media microdrops in 5% CO₂ and        incubated 37° C. overnight.    -   4. Embryos with zonas intact were placed in 0.25% protease in        CMF-HBSS media for 6-8 minutes until the zona had disappeared.    -   5. All embryos were washed 3 times in M2 media after each        treatment step by placing into a fresh drop of M2 media using a        pulled glass capillary tube and syringe. Embryos were split into        experimental and control groups. The experimental embryos        underwent the treatment outlined in steps 6-9; the control        embryos were incubated in M2 media for the equivalent length of        time.    -   6. Zona free day 4.5 mouse embryos were incubated at 37° C. for        1.5 hours in a 50 μl M2 micro-drop containing 5 μl of BioG (50        mg/ml).    -   7. The embryos underwent a second conjugation step where they        were exposed to 5 μl of avidin (1 mg/ml) in a 50 μl micro-drop        of media for 60 minutes at 37° C.    -   8. Finally, a micro-drop was prepared with 50 μl M2 and 25 μl of        BioUE (1 mg/ml). Experimental embryos were placed in the        micro-drop and incubated in for 1 hour at 37° C.

BioUE transformed mouse embryos were subsequently immersed in themodified endometrial cells to test for attachment in a two step process.

-   1. A 4-well culture dish was prepared with 2 wells each containing    50 μl of modified, and stained endometrial cells. Control embryos    and modified embryos were inserted into separate wells and gently    mixed for 30 minutes.-   2. Embryos were carefully removed from the wells and mounted on    glass slides. Embryos were viewed under a fluorescence microscope    and photographed at 200-400× magnification.

The results are outlined in Table 12. TABLE 12 Attachment of murineembryo's inserted with BioG/Av/BioUE to endometrial cells inserted withH type 2 glycolipids (from red cell membranes). Modifications to embryosand endometrial cells Embryo: BioG/Av/BioUE Embryo: nil Endometrialcells: H type 2 + PY Endometrial cells: nil Attachment ++ +/−PY = fluorochrome pyronine YCell attachment scoring = + 1-4 cells, ++ = 5-10 cells, +++ > 10 cellsper embryoND = Not Done

Example 15

The viability of murine embryos following BioG insertion treatment wasconfirmed by continued culture and assessment of morphologicaldevelopment. Eleven 2-Cell mouse embryos underwent BioG insertion withsubsequent wash steps and culture in a 50 μl micro-drop of SQC mediaoverlaid with mineral oil. Sixteen control embryos were cultured in aseparate micro-drop in the same 4-well culture dish (Nunc 176740).Forty-eight hours later there was no difference in morphology betweenthe experimental and control embryos. All embryos had reached theexpected late morula to early blastocyst stage of development. Equalnumbers of embryos initiated zona hatching by Day 5 of culture.

Example 16

The viability of murine embryos treated with biotinylated ganglioside(BioG) was confirmed by the presence of implantation sites and livebirth of pups after embryo transfer (ET) into recipient mice. Theretrieval, treatment, and transfer of embryos were carried out on thesame day at the animal facility. All embryo manipulations, molecularinsertions and incubations were performed in M2 HEPES buffered media ona 37° C. heated microscope stage.

Donor Superovulation and Embryo Retrieval

Large and relatively predictable numbers of embryos can be collected forexperiments by using fertility drugs to stimulate the ovaries ofimmature mice which are highly sensitised to follicle stimulatinghormone (FSH).

Prepubescent (<35 day old) CBA/C57 F1 female mice were injected with 5IUof FSH (Folligon, Pharmaco, NZ) at 1700 and again 48 hours later with5IU of human chorionic gonadotrophin (Pregnyl, Organon, NZ). Each mousewas immediately placed with a CBA male stud mouse of proven fertilityand checked for a seminal plug the following morning. The donors weresacrificed by cervical dislocation on the morning of either Day 1.5 postcoitus for the retrieval of 2-cell embryos or Day 3.5 for late morula toblastocysts. The uterine horns were excised from the abdomen usingsterile technique and placed into a plastic petri dish where they wereflushed with media to expel the embryos.

Embryo BioG Insertion

An equal number of high quality embryos were selected from each donorflushing and pooled together for experimental and control groups.Experimental embryos were placed in a 50 μl micro-drop of M2 media with2.5-5 W of BioG (50 mg/ml) for 1-1.5 hrs at 37° C. The embryos werewashed three times with M2 and placed in a micro-drop of M2 inpreparation for transfer. Control embryos were processed through dropsof media at the same time as experimental embryos.

Embryo Transfer (ET)

To obtain a receptive endometrium in recipient mice, it is necessary tocreate a state of pseudopregnancy by mating with a vasectomised malemouse. The act of coitus rescues the corpus luteum of ovulated folliclesfrom demise and sustains progesterone production necessary forimplantation to occur.

Recipient CBA/C57 F1 female mice in estrus (40-120 days old), wereselected from the pool of mice and placed with a vasectomised male mouseof proven sterility. The time of mating was programmed so thatrecipients were synchronous for 2-Cell embryos transfers or asynchronousby minus 1 day for blastocyst stage transfers. Only recipientsexhibiting a clearly identified seminal plug the following morning wereselected as recipients.

The recipient mice were anaesthetised with 0.8 ml of Avertin (madein-house) and an incision was made in the side of the abdomen above thehip. The fat pad above the ovary was grasped with a serrafin clamp towithdraw the oviduct and uterus outside of the body. Using a 23-28 gaugeneedle, a hole was made in either the bursa of the ovary to expose theinfundibulum for 2-Cell stage transfers, or the uterine horn forblastocyst stage transfers. Six to ten embryos were loaded (using amouth piece) into a fire pulled and polished capillary pipette (approx.150-170 μm in diameter) with mineral oil and air gaps to stabilise theembryos. The pipette was inserted into the prepared needle puncture siteand the embryos expelled until the release of an air-gap was visible.The exposed reproductive tract was replaced into the abdominal cavityand the body wall and skin closed with suture. The mouse was identifiedwith ear marking and observed until conscious.

Mice were housed singularly in cages until they were either sacrificedfor identification of implantation sites or until they had given birth.The implantation (imps) and live birth (pups) rates are presented inTable 13 and 14.

All recipients were kept for 3-6 months post exposure to BioG for healthassessment. The offspring were maintained for breeding of one litter toassess reproductive fitness in the second generation. TABLE 13Implantation rate (imps) and live-birth (pups) outcome of BioG andcontrol embryos. Embryos were zona intact 2-cell embryos. Each transferrepresents a single recipient mouse. Number of embryos Preg. Number ofpups Transfer Treatment Transferred Y/N or imps. (%) Comment I BioG &BioG 6x 2-Cell Y Imps. Each group separated into separate uterine hornsControl Control 6x 2-Cell BioG 2x (33%) Sacrificed day-7 pregnancyControl 2x (33%) II BioG & BioG 5x 2-Cell Y Pups 5x total Same uterinehorn ET Control black mice BioG 3x black (60%) No noticeable differencein anatomy or growth between Different control 5x 2-Cell Control 2x grey(40%) BioG and control mice coloured grey mice ET mum died 1 day beforeweaning - ?stress pups III BioG & BioG 5x 2-Cell Y Pups 5x Same uterinehorn ET Control black mice BioG 4x black (80%) No noticeable differencein anatomy or growth between Different Control 5x 2-Cell Control 1x grey(20%) BioG and control mice coloured grey mice pups IV Control 6x 2-CellY Pups 5x (83%) Born 19 days post ET - normal healthy pups V BioG 10x2-Cell Y Pups 8x (80%) Born 18 days post ET Normal healthy pups VIControl 6x 2-Cell N — — VII BioG 10x 2-Cell N — Died 3 months post ET -unknown cause VIII BioG 10x 2-Cell Y Pups 8x (80%) Born 18 days post ETNormal healthy pups IX BioG 7x 2-Cell Y Pups 6x (85%) Born 18 days postET Normal healthy pups X Control 8x 2-Cell Y Pups 5x (63%) Born 18 dayspost ET Normal healthy pups XI Control 10x 2-Cell Y Pups 6x (60%) Born20 days post ET Normal healthy pups (1x runt)

TABLE 14 Implantation rate (imps %) and live-birth outcome (pups) ofBioG and control embryos. Transfers were into recipient mice at theblastocyst stage zona intact. Day-3.5 embryos were transferred intoday-2.5 (Expt XII-XV) or day-3.5 (Expt. XVI-XVIII) synchronisedrecipient mice. No. embryos Preg No. imps. Transfer TreatmentTransferred Y/N Or pups Comment XII BioG 6x blasts Y Imps 4x (66)%Uterine ET day-2.5 recipient Sacrificed D10 of pregnancy XIII Control 6xblasts Y Imps 6x (100%) Uterine ET day-2.5 recipient Sacrificed D10 ofpregnancy XIV BioG 6x blasts Y Imps 4x (66%) Uterine ET day-2.5recipient Sacrificed D10 of pregnancy XV Control 8x blasts Y Imps 5x(63%) Uterine ET day-2.5 recipient Sacrificed D10 of pregnancy XVIControl 6x blasts Y Pups 4x (66%) Uterine ET day-3.5 Recipients Normalhealthy pups XVII BioG 6x blasts Y Pups 3x (50%) Uterine ET day-3.5Recipients Normal healthy pups XVIII BioG 6x blasts Y Pups 5x (83%)Uterine ET day-3.5 Recipients Normal healthy pups

The first indication that BioG 2-cell embryos were capable ofimplantation was in a recipient mouse that had 6 BioG inserted embryosreplaced into one uterine horn and 6 untreated control embryos replacedinto the other horn (Transfer I). An inspection of the excised uteri onDay 7 of pregnancy revealed 4 implantation sites in each horn.

The second experimental evidence showed that not only were BioG 2-Cellembryos capable of implantation but they also gave rise to live healthypups. In transfers II and III, five embryos derived from a pure blackstrain of mice (C57 donor and stud) were inserted with BioG and replacedinto the same uterine horn as five control embryos derived from a puregrey strain of mouse (CBA donor and stud). The resulting colour of the10 offspring, combined from both recipient mothers, was 3 grey (controlembryos) and 7 black (BioG) babies.

Further ET experiments utilising embryos at two different stages ofdevelopment, 2-cell and blastocyst, revealed similar pregnancy and livebirth rates between BioG embryos and untreated control embryos for bothstages of development. Overall, 8 out of 9 embryo transfers of BioGembryos resulted in a pregnancy with a live birth rate of 72.0%. Thetransfer of control embryos resulted in a pregnancy for 6 out of 7 ETs,with a 72.5% live birth rate.

In conclusion, the insertion of BioG in zona intact embryos from 2-cellto blastocyst stage does not appear to significantly impair theimplantation and ongoing development of the embryo to live birth ofhealthy pups.

Example 17

The viability of zona free and zona intact murine embryos, inserted withbiotinylated ganglioside (BioG) and conjugated sequentially with avidin(Av) and biotinylated IgG was confirmed by the birth of live pups postembryo transfer. Embryo retrieval and transfer of blastocysts wascarried out using the methodology previously described in Example 16.

The data outlined in Table 15 showed similar live birth rates wereobserved for experimental and control treated embryos in both the ZI andZF groups (experimental and control respectively: ZI 61% vs 33%, ZF 83%vs 71.5%). The primary aim of this series of experiments was not tocompare implantation or live birth rates, hence the small numbers andsubsequent lack of statistical analysis. The results do however confirmthat ZI and ZF embryos inserted with the complete BioG/Av/BiolgGmolecule give rise to healthy live pups.

Example 18

The reproductive fitness of experimental offspring and the health of theoffspring proved to be similar to other inbred mice within the sameanimal facility. Offspring from embryo transfer experiments were pairedin cages and allowed to breed. All pairs produced a litter within 75days of birth. The mean size of the litter was 6.2 pups with normalappearance (Table 16). TABLE 16 Number of pups delivered in 1^(st)litter from experimental offspring. Experimental Origin No. of pups inand Pair Treatment 1^(st) litter Example 4 VII BioG 8 Example 4 VIIIBioG 5 Example 4 XI BioG 8 Example 5 I BioG/Av/BioIgG 4 Example 5 IVBioG/Av/BioIgG 7 Example 5 III Control ZI 5 Example 5 V BioG/Av/BioIgG10 Example 5 VI Pronase 6 BioG/Av/BioIgG Example 5 VII Pronase 3BioG/Av/BioIgG

TABLE 15 Pregnancy outcome, number of pups and live birth rate (LB % -number of pups divided by number of embryos transfer) of BioG/Av/BioIgGblastocysts (blasts.) zona intact and zona free. Treatment No. embryosPreg. No. pups Transfer type transferred Y/N (LB %) Comment IBioG/Av/BioIgG 6x blasts Y Pups Born 16 days post ET zona intact 5xNormal healthy pups 83% II Control 6x blasts N — — zona intact IIIControl 6x blasts Y Pups Born 16 days post ET zona intact 2x Normalhealthy pups 33% IV BioG/Av/BioIgG 6x late morula Y Pups Born 17 dayspost ET zona intact 3x Normal healthy pups 50% V BioG/Av/BioIgG 6x latemorula Y Pups Born 17 days post ET zona intact x3 Normal healthy pups50% (1x died) VI Pronase 6x blasts Y Pups Born 16 days post ETBioG/Av/BioIgG zona free 6x Normal healthy pups 100%  (1x died) VIIPronase 6x blasts Y Pups Born 16 days post ET BioG/Av/BioIgG zona free6x Normal healthy pups 100%  VIII Pronase 6x blasts Y Pups Born 17 dayspost ET Control zona free 5x Normal healthy pups 83% IX Pronase 6xblasts Y Pups Born 16 days post ET Control zona free 4x Normal healthypups 66% X Pronase 6x blasts Y Pups Born 16 days post ET BioG/Av/BioIgGzona free 3x Normal healthy pups 50%

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1-101. (canceled)
 102. A method of enhancing the implantation of anembryo into the endometrium of an animal including the steps: a.preparing a modified embryo incorporating a glycolipid-attachmentmolecule construct; and b. transferring the modified embryo to theuterus of the animal.
 103. A method as claimed in claim 102 where theglycolipid-attachment molecule construct comprises an exogenouslymodified glycolipid modified to incorporate a binding part and anattachment molecule modified to incorporate a binding part and where therespective binding parts are adapted to enable the modified glycolipidand the modified attachment molecule to bind to each other eitherdirectly or indirectly through a bridging molecule.
 104. A method asclaimed in claim 103 where the modification to the glycolipid is to thecarbohydrate portion of the glycolipid.
 105. A method as claimed inclaim 103 wherein the attachment molecule is selected from the groupconsisting of: natural or synthetic carbohydrates or oligosaccharides;glycolipids; glycoconjugates; proteins or peptides; acyl groups; andpolymers.
 106. A method as claimed in claim 105 where the attachmentmolecule is selected from the group consisting of: poly L-lysine;antibodies; lectins; polyvinyl pyrrolidine; and functionally equivalentderivatives thereof.
 107. A method as claimed in claim 106 wherein theattachment molecule is an immunoglobulin.
 108. A method as claimed inclaim 107 wherein the attachment molecule is immunoglobulin G (IgG).109. A method as claimed in claim 103 where the attachment molecule isadapted to interact with the epithelial cells of the endometrium, mucus,mucin, or other endogenous or exogenously provided component of mucus.110. A method as claimed in claim 109 where the attachment molecule isan endometrial attachment molecule.
 111. A method as claimed in claim103 where the glycolipid is selected from the group consisting ofphosphoglycerides and sphingolipids.
 112. A method as claimed in claim103 where the attachment molecule and the glycolipid are bound togetherby simple non-covalent binding interactions including ionic, van deWaals, water exclusion, electrostatic, hydrogen bonding and chelationbinding.
 113. A method as claimed in claim 103 where the attachmentmolecule and the glycolipid are bound together by covalent bonding. 114.A method as claimed in claim 103 where the attachment molecule and theglycolipid are bound together by avidin-biotin binding.
 115. A method asclaimed in claim 114 where the binding part of the glycolipid comprisesbiotin and the binding part of the attachment molecule comprises avidin.116. A method as claimed in claim 114 where the binding part of theglycolipid comprises avidin and the binding part of the attachmentmolecule comprises biotin.
 117. A method as claimed in claim 114 wherethe attachment molecules and the glycolipid are bound together through abridging molecule.
 118. A method as claimed in claim 117 where thebridging molecule comprises avidin and the binding part of both theattachment molecule an the glycolipid comprises biotin.
 119. A method asclaimed in claim 117 wherein the bridging molecule comprises biotin andthe binding part of both the attachment molecule and the glycolipidcomprises avidin.
 120. A method as claimed in claim 103 where theattachment molecule and the glycolipid are bound together by a chelationinteration between at least one chelator and a chelated metal ion. 121.A method as claimed in claim 120 wherein the binding part of both theattachment molecule and the glycolipid comprises a chelator.
 122. Amethod as claimed in claim 120 wherein the chelator is a poly-histidinerecombinant protein.
 123. A method as claimed in claim 120 where thechelator is attached covalently to the glycolipid.
 124. A method asclaimed in claim 120 where the chelator is attached non-covalently tothe glycolipid.
 125. A method as claimed in claim 124 wherein thechelator is attached to the glycolipid via biotin or avidin.
 126. Amethod as claimed in claim 120 where the chelated metal ion is Co²⁺,Ni²⁺ or Cu²⁺.
 127. A method as claimed in claim 103 where the glycolipidmodified to incorporate a binding part is a biotinylated glycolipid.128. A method as claimed in claim 103 where the glycolipid of theganglioside class that contains sialic acid groups, or a glycolipid ofthe neutral class that contains galactose.
 129. A method as claimed inclaim 103 where the attachment molecule is a molecule that has a bindingaffinity for molecules on cell membranes including the mucus coat ofcell membranes.
 130. A method as claimed in claim 129 wherein themolecules on cell membranes are receptor sites and/or blood grouprelated antigens.
 131. A method as claimed in claim 130 where the cellmembranes are endometrial.
 132. A method as claimed in claim 102 wherethe animal is a human or domesticated animal.
 133. A method as claimedin claim 102 where the modified embryo is prepared from a species,hybrid or variety of animal different from the species, hybrid orvariety of animal of the uterus.